In continuation of our attempts to elucidate the profound hypothermic effect of nucleotides and nucleosides we have measured plasma levels of adenosine and inosine plasma levels during exogenous administration of adenosine, AMP, and ATP. Baseline plasma concentrations of adenosine averaged 311 22 pmol/ml (n =7) and of inosine 302 56 pmol/ml (n = 7). Plasma concentrations of adenosine 40 min after injection of 1.5 &#61549;mol/g of ATP, AMP, or adenosine increased markedly to 1325 115 pmol/ml (n=5), 1629 221 pmol/ml (n=6), and 2387 155 pmol/ml (n=6) while plasma concentrations of inosine after ATP, AMP or adenosine injection increased to even higher levels of 9894 2472 pmol/ml (n=5), 15213 4279 pmol/ml (n=6), and 22876 3904 pmol/ml (n=4). Thus, adenosine and inosine plasma concentrations were significantly elevated over baseline by all injected nucleotides although adenosine plasma levels resulting from adenosine injections were significantly higher than those caused by ATP and AMP injections. We conclude from these data that the hypothermic effects of AMP and ATP could conceivably be caused by adenosine since the nucleotides are effectively converted to adenosine. To identify the adenosine receptor subtype involved in causing adenosine-induced hypothermia we assessed the effects of CPA (n = 5), CGS 21680 (n = 5) and IB-MECA (n = 5) on core body temperature. The A1AR agonist CPA decreased temperature by about 0.9 0.2 C to 37.7 0.3 C at 0.1 mg/kg bw, by about 4.0 0.5 C to 32.8 0.5 C at 0.5 mg/kg bw, and by about 12.2 0.2 C to 26.3 0.3 C at 1.0 mg/kg bw (all p <0.05 compared to baseline). Consistent with a specific A1AR-induced hypothermia is our observation that in A1AR-deficient mice CPA (1 mg/kg bw;n = 5) caused only a short-lasting temperature decrease (by 2.4 0.3 C). In contrast to the marked hypothermia caused by A1AR activation, the hypothermic effects of the A2aAR agonist CGS 21680 (temperature reduction of 2.9 0.2 C at 1.0 mg/kg body weight) and of the A3AR agonist IB-MECA (temperature reduction of 0.9 0.1 C at 1.0 mg/kg body weight) were significantly less pronounced (all p <0.05 compared to CPA at same dose). The conclusion that only the A1AR subtype is a potential mediator of adenosine-induced hypothermia was corroborated by studies in A1AR-deficient mice. In wild type mice, adenosine at doses of 0.15, 0.75, and 1.5 &#61549;mol/g bw caused a maximal reduction of core body temperature by 1.4 0.2 C, 7.9 1.1 C, and 12.4 0.3 C. In A1AR-/- mice these responses were reduced to 0.5 0.3 C, 4.9 0.5 C (p<0.05 compared to wt), and 7.3 0.7 C (p<0.05 compared to wild type). Thus, about 44% of the total hypothermic response to adenosine at the intermediate dose and 35% at the high dose appear to be mediated by A1AR surface receptors. Because the administration of ATP and AMP caused a marked increase of plasma adenosine we also tested the effect of these nucleotides on hypothermia in A1AR-/- animals. Unexpectedly, the hypothermia caused by both ATP and AMP in the absence of A1AR was attenuated to a similar degree as that caused by adenosine. At doses of 0.15, 0.75, and 1.5 &#61549;mol/g body weight, ATP and AMP induced a maximal CBT reduction of 0.6 0.3 C and 2.16 0.5 C, 4.4 0.4 C and 7.9 1.1 C, and 10.2 0.6 C and 10.8 0.7 C in wt mice. In A1AR-/- mice, maximal CBT reduction caused by ATP and AMP were only 0.1 0.1 C and 0.7 0.3 C, 1.0 0.4 C and 4.2 0.6 C (p<0.05 compared to wt), and 6.4 0.9 C and 6.6 0.5 C (p<0.05 compared to wt). Remarkably, the apparent relative contribution of A1AR to the hypothermia caused by ATP and AMP was nearly identical to their contribution to adenosine-induced hypothermia. An important contribution of A1AR to the hypothermic effect of AMP is further supported by the finding that the A1AR specific inhibitor DPCPX significantly reduced AMP hypothermia. In wild type mice pretreated with DPCPX, AMP at 1.5 &#61549;mol/g reduced body temperature by 5.2 0.4 C (are under the curve: 409 50 Cmin;n = 5) whereas in the absence of DPCPX the AMP-induced body temperature reduction was 7.6 0.4 C (area under the curve: 661 99 Cmin;n = 5;p <0.05 compared to DPCPX). To assess a potential role of AMP-activated kinase (AMPK) in the hypothermia induced by AMP we determined the effect of the AMPK activator AICAR. AICAR in a dose equimolar to the highest AMP dose (1.5 &#61549;mol/g) did not affect CBT (n = 3). In a much higher dose (1 mg/g) that has previously been shown to activate AMPK, AICAR decreased CBT by 1.8 0.5 C to 36.6 0.4 C (n = 5;p <0.05) at 60 min after injection, and thus did not induce overt hypothermia. Since only part of the hypothermic effect of adenosine appears to be mediated by A1AR surface receptors we investigated a possible role of intracellular adenosine on core body temperature. Low concentrations of cytosolic adenosine concentrations are achieved predominantly through efficient phosphorylation of adenosine by adenosine kinase (AK), a strictly intracellular enzyme. Thus, in the present studies we determined the hypothermic effect of the AK inhibitor A-134974. In fact, inhibition of AK was associated with a long lasting and profound hypothermia with a nadir of 31.2 0.7 C and an overall effect expressed as area under the curve of 2110 218 Cmin (n = 5). It is important to note that A1AR surface receptors cannot have contributed to this effect since the experiments were performed in A1AR-/- mice. In contrast, during inhibition of adenosine breakdown to inosine by the adenosine deaminase inhibitor EHNA, there was a slight decrease in body temperature from 38.3 0.2 C to 36.9 0.5 C, but temperature did not drop below the hypothermic border of 36 C. The drop in CBT caused by the AK inhibitor A-134974 was accompanied by a rise of the plasma levels of adenosine from 145 30 pmol/ml to 663 25 pmol/ml (n = 5;p<0.05) and of inosine from 193 52 pmol/ml to 1058 156 pmol/ml (n=5;p <0.05). The increase of plasma adenosine, presumably the result of transport of adenosine to the extracellular space driven by increased intracellular concentrations, was 2 to 3.6-fold lower than the adenosine levels of wild type mice after injection of ATP, AMP or adenosine. We conclude that adenosine causes hypothermia in part through mediation of A1AR surface receptors, but that another part of the overall effect is the result of an elevation of intracellular adenosine levels. AMP and ATP exert their effect mostly by conversion to adenosine. The mechanisms of action of extracellular and intracellular adenosine in affecting cellular metabolism need to be further defined. The usefulness of adenosine-induced hypothermia as an approach to reduce metabolism, for example during surgery, may be considered.